Regulatory agencies around the world place stringent requirements on commercial manufacturers of biopharmaceutical compounds to provide biosafety assurance of their drugs. The manufacturers have to build in and validate at least two orthogonal steps, i.e. operating by two distinct mechanisms, of virus removal into their processes, one of which is usually size-based filtration. The combined LRV (log reduction value) for retrovirus removal of all removal steps of the combined purification process must be at least 17, of which filtration provides at least 6.
Retrovirus is a type of RNA virus (such as HIV) that reproduces by transcribing itself into DNA (using reverse transcriptase). The resultant DNA inserts itself into a cell's DNA and is reproduced by the cell. Two particularly dangerous retroviruses to humans are Human Immunodeficiency Virus (HIV) and Human T-Cell Leukemia Virus (HTLV). The size of retroviruses falls in the range from about 80 to 130 nm. In development of virus-retentive filtration products, it is common practice to substitute mammalian viruses for bacteriophages of similar size, shape, and surface charge. Examples of such bacteriophages include Phi-6 and PR772. Practice shows that filters exhibiting a certain level of retention with these bacteriophages usually exhibit same or higher levels of retention with mammalian retroviruses.
A detailed description of industry approaches to virus removal and a list of commercially available virus removal products is available in “Filtration and Purification in the Biopharmaceutical Industry” by T. Meltzer and M. Jornitz, eds., 2nd edition, Informa Healthcare USA, Inc., 2008, Chapter 20, pp. 543-577.
There are a number of commercially available membranes validated for retrovirus removal. A typical retrovirus removal membrane, for example the Retropore® available from Millipore Corporation of Billerica, Mass. The membrane has been extensively tested using the 78 nm diameter bacteriophage Phi 6. This bacteriophage is readily grown to monodispersed, uniform size, and high titer challenges. A consistent >6.5 LRV has been observed over the range of feedstock and processing conditions. The Retropore® membrane is manufactured according to U.S. Pat. No. 7,108,791, which is fully incorporated herein by reference. The Retropore® membrane has an asymmetrical pore structure, with a tight virus removal side and microporous “support” side, and is manufactured by a traditional phase inversion process used to make a wide range of UF and MF membranes.
One of the inherent limitations of this process is that porosity decreases with decrease in pore size. For example, a microporous membrane with average pore size of about 0.5 micron may have a porosity about 75% to 80%, while an ultrafiltration membrane having an average pore size of about 0.01 micron to 0.02 micron will only be about 5% to 30% porous in its region of narrowest pore size. Retrovirus removal membranes have traditionally low porosity and thus lower flux. U.S. Pat. No. 7,108,791 defines minimum desirable flux of a “large virus” (>75 nm) filter as having a minimum value of 5 to 20 lmh/psi.
U.S. Pat. No. 7,459,085 to Asahi Kasei Medical Co., Ltd., discloses a hydrophilic microporous membrane comprising a thermoplastic resin having a maximum pore size of 0 to 100 nm and designed for low protein fouling in virus filtration application.
Published US Pat. App. 2008/0004205 to Millipore Corp. discloses an integral multilayered composite membrane having at least one ultrafiltration layer designed for virus removal ultrafiltration membranes and methods of making such membrane.
As biopharmaceutical manufacturing becoming more mature, the industry is constantly looking for ways to streamline the operations, combine and eliminate steps, and dramatically reduce the time it takes to process each batch of the drug. At the same time, there are market and regulatory pressures requiring manufacturers to reduce their costs. Since virus filtration accounts for a significant percentage of the total cost of drug purification, any approaches to increase membrane throughput and reduce time are valuable. With the introduction of new prefiltration media and corresponding increase in throughput of virus filters, filtration of more and more feed streams is becoming flux-limited. Thus, dramatic improvements in the permeability of virus filters will have a direct effect on the cost of virus filtration step.
Filters used in liquid filtration can be generally categorized as either fibrous nonwoven media filters or porous film membrane filters.
Fibrous nonwoven liquid filtration media include, but are not limited to, nonwoven media formed from spunbonded, melt blown or spunlaced continuous fibers; hydroentangled nonwoven media formed from carded staple fiber and the like; or some combination of these types. Typically, fibrous nonwoven filter media filters used in liquid filtration have pore sizes generally greater than about 1 micron (μm).
Porous film membrane liquid filtration media is used either unsupported or used in conjunction with a porous substrate or support. Porous filtration membranes have pore sizes smaller than the fibrous nonwoven media, and typically have pore sizes less than about 1 μm. Porous film liquid filtration membranes can be used in: (a) microfiltration, wherein particulates filtered from a liquid are typically in the range of about 0.1 μm to about 10 μm; (b) ultrafiltration, wherein particulates filtered from a liquid, are typically in the range of about 5 nm to about 0.1 μm; and (c) reverse osmosis, wherein particulate matter filtered from a liquid, are typically in the range of about 1 Å to about 1 nm. Retrovirus-retentive membranes are usually considered to be on the open end of ultrafiltration membranes.
The two most desired features of a liquid membrane are high permeability and reliable retention. Naturally, there is a trade-off between these two parameters, and for the same type of membrane, greater retention can be achieved by sacrificing permeability of the membrane. The inherent limitations of the conventional processes for making porous film membranes prevent membranes from exceeding a certain threshold in porosity, and thus limits the magnitude of permeability that can be achieved at a given pore size.
Electrospun nanofiber mats are highly porous polymeric materials, wherein the “pore” size is linearly proportional to the fiber diameter, while the porosity is relatively independent of the fiber diameter. The porosity of electrospun nanofiber mats usually falls in the range of about 85% to 90%, and resulting in nanofiber mats demonstrating dramatically improved permeability as compared to immersion cast membranes having a similar thickness and pore size rating. Moreover, this advantage becomes amplified in the smaller pore size range, such as that typically required for virus filtration, because of the reduced porosity of UF membranes discussed supra.
The random nature of electrospun mat formation has led to the general assumption that they are unsuitable for any critical filtration of liquid streams. Electrospun nanofiber mats are often referred to as “non-wovens”, thus placing them in the same category with melt-blown and spunbonded fibrous media, what is called “traditional” non-wovens.
Fibers in traditional non-wovens are usually at least about 1,000 nm in diameter, so their effective pore sizes are always more than about one micron. Also, the methods of manufacturing of traditional non-wovens lead to highly inhomogeneous fiber mats, which limits their applicability to liquid filtration.
Synthetic polymers have been formed into webs of very small diameter fibers, (i.e., on the order of a few micrometers or less than 1 μm), using various processes including melt blowing, electrostatic spinning and electroblowing. Such webs have been shown to be useful as liquid barrier materials and filters. Often they are combined with stronger sheets to form composites, wherein the stronger sheets provide the strength to meet the needs of the final filter product.
U.S. Pat. No. 7,585,437 to Jirsak teaches a nozzle-free method for producing nanofibres from a polymer solution using electrostatic spinning and a device for carrying out the method.
WIPO patent application no. WO/2003/080905, “A Manufacturing Device And Method of Preparing For The Nanofibers Via Electro-Blown Spinning Process”, assigned to Nano Technics Co. LTD., and incorporated herein by reference in its entirety, teaches an electroblowing process, wherein stream of polymeric solution comprising a polymer and a solvent is fed from a storage tank to a series of spinning nozzles within a spinneret, to which a high voltage is applied and through which the polymeric solution is discharged. Meanwhile, compressed air, that may optionally be heated, is release from air nozzles disposed in the sides of, or at the periphery of the spinning nozzle. The air is directed generally downward as a blowing gas stream which envelopes and forwards the newly issued polymeric solution and aids in the formation of the fibrous web, which is collected on a grounded porous collection belt above a vacuum chamber. The electroblowing process permits formation of commercial sizes and quantities of nanowebs at basis weights in excess of about 1 gsm to great than about 40 gsm, in a relatively short time period.
U.S. Patent Publication No. 2004/0038014 issued to Schaefer et al. teaches a nonwoven filtration mat comprising one or more layers of a thick collection of fine polymeric microfibers and nanofibers formed by electrostatic spinning for filtering contaminants. The electrostatic spinning process utilizes an electro spinning apparatus including a reservoir in which the fine fiber forming polymer solution is contained, a pump and an emitter device which obtains polymer solution from the reservoir. In the electrostatic field, a droplet of the polymer solution is accelerated by the electrostatic field toward a collecting media substrate located on a grid. A high voltage electrostatic potential is maintained between the emitter and grid, with the collection substrate positioned there between, by means of a suitable electrostatic voltage source.
U.S. Patent Publication No. 2007/0075015 issued to Bates et al. teaches a liquid filtration media including at least one layer of nanofibers having average diameters less than 1,000 nanometers optionally disposed on scrim layer for filtering particulate matter in a liquid. The filtration media have flow rates of at least 0.055 L/min/cm2 at relatively high levels of solidity. The media apparently has non-diminishing flow rates as differential pressures increase between 2 psi (14 kPa) and 15 psi (100 kPa).
U.S. Patent Publication No. 2009/0026137 issued to Chen teaches fabricating liquid filter with a composite medium that has a nanoweb adjacent to and optionally bonded to a microporous membrane. The membrane is characterized by an LRV value of 3.7 at a rated particle size, and the nanoweb has a fractional filtration efficiency of greater than 0.1 at the rated particle size of the membrane. The nanoweb also has a thickness efficiency ratio of greater than 0.0002 at that efficiency. The nanoweb acts to provide depth filtration to the membrane.
U.S. Pat. No. 7,144,533 to Koslow teaches s nanofiber mats coated with microbiological interception enhancing agent (such as a cationic metal complex) that provide greater than 4 LRV of viral removal and 6 LRV of bacterial removal.
U.S. Patent Publication No. 2009/0199717 to Green teaches a method to form an electrospun fiber layer carried by the substrate layer, the fine fiber layer including a significant amount of fibers with a diameter of less than 100 nanometers.
Bjorge et al. in Desalination 249 (2009) 942-948 teach electrospun Nylon nanofiber mats of 50-100 nm diameter and 120 um thickness. The measured bacteria LRV for non-surface treated fibers is 1.6-2.2. The authors conclude that bacteria removal efficiency of as-spun nanofiber mats is unsatisfactory.
Gopal et al. in Journal of Membrane Science 289 (2007) 210-219, teach electrospun polyethersulfone nanofiber mats, wherein the nanofibers have a diameter of about 470 nm, such that the during liquid filtration the mats act as a screen to filter our particles above 1 micron and as a depth filter for particles under 1 micron.
D. Aussawasathien et al. in Journal of Membrane Science, 315 (2008) 11-19, teach electrospun nanofibers of 30-110 nm diameter used in removal of polystyrene particles (0.5-10 um diameter).
It would be desirable to have a reliable electrospun nanofiber filter medium suitable for >99.9999% (LRV>6) removal of retroviral particles, while simultaneously achieving high permeability. These nanofiber mats would have three advantages over traditionally used virus removal membranes: (1) higher permeability as a result of higher porosity, (2) free-standing nature, (i.e., no supporting microporous structure is required), and (3) potential to be used in single layer format. The latter two advantages offer considerably greater flexibility in the design and validation of virus filtration devices.
Additionally, the porous electrospun nanofiber filtration medium would be readily scalable, adaptable to processing volumes of sample fluids ranging from milliliters to thousands of liters, and capable of use with a variety of filtration processes and devices. The invention is directed to these, as well as other objectives and embodiments.